Therapeutic and prophylactic methods using heat shock proteins

ABSTRACT

The present invention relates to immunogenic complexes of heat shock proteins (hsp) noncovalently bound to exogenous antigenic molecules which when administered to an individual elicit specific immunological responses in the host. Methods of prevention and treatment of cancer and infectious disease are provided.

This is a division of application Ser. No. 08/527,547, filed Sep. 13,1995 now U.S. Pat. No. 5,935,576, which is incorporated by referenceherein in its entirety.

This invention was made with government support under grant numberCA44786 awarded by the National Institutes of Health. The government hascertain rights in the invention.

1. INTRODUCTION

The present invention relates to compositions for the prevention andtreatment of primary and metastatic cancers and/or infectious diseases.In the practice of the preventive and therapeutic methods of theinvention, compositions of noncovalent complexes of heat shock/stressproteins (hsp) including, but not limited to, hsp70, hsp90, gp96 aloneor in combination with each other, and antigenic molecules are used toaugment the immune responses to genotoxic and nongenotoxic factors,tumors, pathogens and infectious agents.

2. BACKGROUND OF THE INVENTION

Studies on the cellular response to heat shock and other physiologicalstresses have identified important families of proteins that areinvolved not only in cellular protection against these aggressions, butalso in essential biochemical and immunological processes in unstressedcells. The heat shock proteins include, but are not limited to hsp70,hsp90, gp96, and hsp100; these hsp families accomplish different kindsof chaperoning functions. For example, hsp70, located in the cellcytoplasm, nucleus, mitochondria, or endoplasmic reticulum, (Lindquist,S., et al., 1988, Ann. Rev. Genetics 22:631-677) are involved in thepresentation of antigens to the cells of the immune system, and are alsoinvolved in the transfer, folding and assembly of proteins in normalcells. Similarly, Hsp90 located in the cytosol are involved inchaperoning and gp96 present in the endoplasmic reticulum are involvedin antigen presentation (Srivastava, P. K., et al., 1991, Curr. Topicsin Microbiology & Immun. 167:109-123).

2.1. Immunotherapy

In modern medicine, immunotherapy or vaccination has virtuallyeradicated diseases such as polio, tetanus, tuberculosis, chicken pox,measles, hepatitis, etc. The approach using vaccinations has exploitedthe ability of the immune system to prevent infectious diseases. Suchvaccination with non-live materials such as proteins generally leads toan antibody response or CD4+ helper T cell response. Raychaudhuri, S.and Morrow, W. J. W., 1993, Immunology Today, 14:344-348. On the otherhand, vaccination or infection with live materials such as live cells orinfectious viruses generally leads to a CD8+ cytotoxic T-lymphocyte(CTL) response. A CTL response is crucial for protection againstcancers, infectious viruses and bacteria. This poses a practicalproblem, for, the only way to achieve a CTL response is to use liveagents which are themselves pathogenic. The problem is generallycircumvented by using attenuated viral and bacterial strains or bykilling whole cells which can be used for vaccination. These strategieshave worked well but the use of attenuated strains always carries therisk that the attenuated agent may recombine genetically with host DNAand turn into a virulent strain. Thus, there is need for methods whichcan lead to CD8+ CTL response by vaccination with non-live materialssuch as proteins in a specific manner.

The era of tumor immunology began with experiments by Prehn and Main,who showed that antigens on the methylcholanthrene (MCA)-inducedsarcomas were tumor specific in that transplantation assays could notdetect these antigens in normal tissue of the mice (Prehn, R. T., etal., 1957, J. Natl. Cancer Inst. 18:769-778). This notion was confirmedby further experiments demonstrating that tumor specific resistanceagainst MCA-induced tumors can be elicited in the autochthonous host,that is, the mouse in which the tumor originated (Klein, G., et al.,1960, Cancer Res. 20:1561-1572).

In subsequent studies, tumor specific antigens were also found on tumorsinduced with other chemical or physical carcinogens or on spontaneoustumors (Kripke, M. L., 1974, J. Natl. Cancer Inst. 53:1333-1336; Vaage,J., 1968, Cancer Res. 28:2477-2483; Carswell, E. A., et al., 1970, J.Natl. Cancer Inst. 44:1281-1288). Since these studies used protectiveimmunity against the growth of transplanted tumors as the criterion fortumor specific antigens, these antigens are also commonly referred to as“tumor specific transplantation antigens” or “tumor specific rejectionantigens.” Several factors can greatly influence the immunogenicity ofthe tumor induced, including, for example, the specific type ofcarcinogen involved, immunocompetence of the host and latency period(Old, L. J., et al., 1962, Ann. N.Y. Acad. Sci. 101:80-106; Bartlett, G.L., 1972, J. Natl. Cancer Inst. 49:493-504).

Most, if not all, carcinogens are mutagens which may cause mutation,leading to the expression of tumor specific antigens (Ames, B. N., 1979,Science 204:587-593; Weisburger, J. H., et al., 1981, Science214:401-407). Some carcinogens are immunosuppressive (Malmgren, R. A.,et al., 1952, Proc. Soc. Exp. Biol. Med. 79:484-488). Experimentalevidence suggests that there is a constant inverse correlation betweenimmunogenicity of a tumor and latency period (time between exposure tocarcinogen and tumor appearance) (Old, L. J., et al., 1962, Ann. N.Y.Acad. Sci. 101:80-106; and Bartlett, G. L., 1972, J. Natl. Cancer Inst.49:493-504). Other studies have revealed the existence of tumor specificantigens that do not lead to rejection, but, nevertheless, canpotentially stimulate specific immune responses (Roitt, I., Brostoff, Jand Male, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pp.17.1-17.12).

2.2. Tumor-specific Immunogenicities of Heat Shock/Stress Proteinshsp70, hsp90 and gp96

Srivastava et al. demonstrated immune response tomethylcholanthrene-induced sarcomas of inbred mice (1988, Immunol. Today9:78-83). In these studies it was found that the molecules responsiblefor the individually distinct immunogenicity of these tumors wereidentified as cell-surface glycoproteins of 96 kDa (gp96) andintracellular proteins of 84 to 86 kDa (Srivastava, P. K., et al., 1986,Proc. Natl. Acad. Sci. USA 83:3407-3411; Ullrich, S. J., et al., 1986,Proc. Natl. Acad. Sci. USA 83:3121-3125. Immunization of mice with gp96or p84/86 isolated from a particular tumor rendered the mice immune tothat particular tumor, but not to antigenically distinct tumors.Isolation and characterization of genes encoding gp96 and p84/86revealed significant homology between them, and showed that gp96 andp84/86 were, respectively, the endoplasmic reticular and cytosoliccounterparts of the same heat shock proteins (Srivastava, P. K., et al.,1988, Immunogenetics 28:205-207; Srivastava, P. K., et al., 1991, Curr.Top. Microbiol. Immunol. 167:109-123). Further, hsp70 was shown toelicit immunity to the tumor from which it was isolated but not toantigenically distinct tumors. However, hsp70 depleted of peptides wasfound to lose its immunogenic activity (Udono, M., and Srivastava, P.K., 1993, J. Exp. Med. 178:1391-1396). These observations suggested thatthe heat shock proteins are not immunogenic per se, but are carriers ofantigenic peptides that elicit specific immunity to cancers (Srivastava,P. K., 1993, Adv. Cancer Res. 62:153-177).

2.3. Pathobiology of Cancer

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia.

Pre-malignant abnormal cell growth is exemplified by hyperplasia,metaplasia, or most particularly, dysplasia (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W.B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

The neoplastic lesion may evolve clonally and develop an increasingcapacity for invasion, growth, metastasis, and heterogeneity, especiallyunder conditions in which the neoplastic cells escape the host's immunesurveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rded., Mosby, St. Louis, pps. 17.1-17.12).

3. SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions, methods, andkits for prevention and treatment of cancer and/or infectious diseasesby enhancing the host's immunocompetence and activity of immune effectorcells. The pharmaceutical compositions of the invention comprisecomplexes of hsps noncovalently bound to exogenous antigenic molecules.The exogenous antigenic molecules differ from the peptides endogenouslycomplexed with hsps in vivo and which copurify with the hsps. Theexogenous antigenic molecules are antigens/immunogens orantigenic/immunogenic fragments or derivatives thereof. Such antigenicmolecules can be selected from among those known in the art or assayedby the ability to bind to antibody or MHC molecule (antigenicity) orgenerate immune response (immunogenicity) by standard immunoassays knownin the art. The antigenic molecules are noncovalently complexed withhsps in vitro prior to administration to a patient. For treatment orprevention of cancer, the antigenic molecules are molecules that willinduce an immune response against the cancer, e.g., tumor-specificantigens, or tumor-associated antigens, preferably of human tumors. Fortreatment or prevention of infectious diseases, the antigenic moleculesare molecules that will induce an immune response against the infectiousagent, e.g., antigens of viruses, bacteria, fungi, parasites etc.,preferably agents that infect humans. In a specific embodiment, thepharmaceutical compositions of the present invention include hspscomplexed not only to a single antigen but also more than one antigen oran entire cocktail of (e.g., tumor specific) antigens. Preferably, thepatient is a human, and the hsps are human hsps. The hsps in thecomplexes can be autologous or allogeneic to the patient.

Particular compositions of the invention and their properties aredescribed in the sections and subsections which follow. Preferably, thecomplex of hsp and antigenic molecules comprises hsp70, hsp90, gp96, ora combination thereof.

In another embodiment, the pharmaceutical compositions further compriseeffective amounts of a biological response modifier, including but notlimited to the cytokines interferon-α (IFN-α), IFN-γ, interleukin-2(IL-2), IL-4, IL-6, tumor necrosis factor (TNF), or other cytokinegrowth factor.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of administration of hsp70 complexed with ovalbumin orcytotoxicity of T cells against the EG7 cell line (expresses ovalbuminantigen) or the EL4 cell line (negative for ovalbumin antigen).

FIG. 1A: Two mice in each group were immunized with: a) a controlvehicle (squares); b) ovalbumin alone (plus sign); c) hsp70 alone(triangles); or d) hsp70-ovalbumin complex. T cells taken from theimmunized mice were tested for cytotoxicity against the EG7 cells (FIG.1A) or EL4 cells (FIG. 1B). The results demonstrate that thehsp70-ovalbumin complex is a far better reagent at inducing a cytotoxicT-lymphocyte response than ovalbumin alone or hsp70 alone (FIG. 1A). TheT cells did not respond in the presence of the EL4 cells which lack theovalbumin antigen (FIG. 1B).

5. DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for the prevention and treatment of primary andmetastatic cancers and/or infectious diseases are described. Theinvention provides pharmaceutical compositions of hsp noncovalentlybound to exogenous antigenic molecules.

The exogenous antigenic molecules differ from the peptides endogenouslycomplexed with hsps in vivo and which copurify with the hsps. Theexogenous antigenic molecules are antigens/immunogens orantigenic/immunogenic fragments or derivatives thereof. Such antigenicmolecules can be selected from among those known in the art or assayedby the ability to bind to antibody or MHC molecule (antigenicity) orgenerate immune response (immunogenicity) by standard immunoassays knownin the art. The antigenic molecules are noncovalently complexed withhsps in vitro prior to administration to a patient. For treatment orprevention of cancer, the antigenic molecules are molecules that willinduce an immune response against the cancer, e.g., tumor-specificantigens, or tumor-associated antigens, preferably of human tumors. Fortreatment or prevention of infectious diseases, the antigenic moleculesare molecules that will induce an immune response against the infectiousagent, e.g., antigens of viruses, bacteria, fungi, parasites etc.,preferably agents that infect humans. In a specific embodiment, thepharmaceutical compositions of the present invention include hspscomplexed not only to a single antigen but also more than one antigen oran entire cocktail of (e.g., tumor specific) antigens. Preferably, thepatient is a human, and the hsps are human hsps. The hsps in thecomplexes can be autologous or allogeneic to the patient.

The methods of the invention comprise methods of eliciting an immuneresponse in an individual in whom the treatment or prevention ofinfectious diseases or cancer is desired by administering a compositioncomprising an effective amount of a complex, in which the complexconsists essentially of a hsp noncovalently bound to an exogenousantigenic molecule. The hsp and/or the antigenic molecule can beisolated from the individual or from others or by recombinant productionmethods using a cloned hsp originally derived from the individual orfrom others. Exogenous antigens and fragments and derivatives (bothpeptide and non-peptide) thereof for use in complexing with hsps, can beselected from among those known in the art, as well as those readilyidentified by standard immunoassays know in the art by the ability tobind antibody or MHC molecules (antigenicity) or generate immuneresponses (immunogenicity).

The hsps of the present invention that can be used include but are notlimited to, hsp70, hsp90, gp96 alone or in combination. Preferably, thehsps are human hsps.

Heat shock proteins, which are also referred to interchangeably hereinas stress proteins, useful in the practice of the instant invention canbe selected from among any cellular protein that satisfies any one ofthe following criteria. It is a protein whose intracellularconcentration increases when a cell is exposed to a stressful stimuli,it is capable of binding other proteins or peptides, and it is capableof releasing the bound proteins or peptides in the presence of adenosinetriphosphate (ATP) or low pH; or it is a protein showing at least 35%homology with any cellular protein having any of the above properties.

The first stress proteins to be identified were the heat shock proteins(hsps). As their name implies, hsps are synthesized by a cell inresponse to heat shock. To date, three major families of hsp have beenidentified based on molecular weight. The families have been calledhsp60, hsp70 and hsp90 where the numbers reflect the approximatemolecular weight of the stress proteins in kilodaltons. Many members ofthese families were found subsequently to be induced in response toother stressful stimuli including, but not limited to, nutrientdeprivation, metabolic disruption, oxygen radicals, and infection withintracellular pathogens. (See Welch, May 1993, Scientific American56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420; Craig, 1993, Science260:1902-1903; Gething, et al., 1992, Nature 355:33-45; and Lindquist,et al., 1988, Annu. Rev. Genetics 22:631-677), the disclosures of whichare incorporated herein by reference.

It is contemplated that hsps/stress proteins belonging to all of thesethree families can be used in the practice of the instant invention.

The major hsps can accumulate to very high levels in stressed cells, butthey occur at low to moderate levels in cells that have been stressed.For example, the highly inducible mammalian hsp70 is hardly detectableat normal temperatures but becomes one of the most actively synthesizedproteins in the cell upon heat shock (Welch, et al., 1985, J. Cell.Biol. 101:1198-1211). In contrast, hsp90 and hsp60 proteins are abundantat normal temperatures in most, but not all, mammalian cells and arefurther induced by heat (Lai, et al., 1984, Mol. Cell. Biol. 4:2802-10;van Bergen en Henegouwen, et al., 1987, Genes Dev. 1:525-31).

Heat shock proteins are among the most highly conserved proteins inexistence. For example, DnaK, the hsp70 from E. coli has about 50% aminoacid sequence identity with hsp70 proteins from excoriates (Bardwell, etal., 1984, Proc. Natl. Acad. Sci. 81:848-852). The hsp60 and hsp90families also show similarly high levels of intra families conservation(Hickey, et al., 1989, Mol. Cell. Biol. 9:2615-2626; Jindal, 1989, Mol.Cell. Biol. 9:2279-2283). In addition, it has been discovered that thehsp60, hsp70 and hsp90 families are composed of proteins that arerelated to the stress proteins in sequence, for example, having greaterthan 35% amino acid identity, but whose expression levels are notaltered by stress. Therefore it is contemplated that the definition ofstress protein, as used herein, embraces other proteins, muteins,analogs, and variants thereof having at least 35% to 55%, preferably 55%to 75%, and most preferably 75% to 85% amino acid identity with membersof the three families whose expression levels in a cell are enhanced inresponse to a stressful stimulus. The purification of stress proteinsbelonging to these three families is described below.

The immunogenic complexes of hsp and exogenous antigenic molecules ofthe invention include any complex containing an hsp and an exogenousantigenic molecule that is capable of inducing an immune response in amammal. The antigenic molecules are noncovalently associated with thehsps. Preferred complexes comprise hsp60, hsp70, or hsp90, noncovalentlybound to a protein antigen. In a specific embodiment, the complexcomprises an hsp called gp96 which is present in the endoplasmicreticulum of eukaryotic cells and is related to the cytoplasmic hsp90s.

Although the hsps can be allogeneic to the patient, in a preferredembodiment, the hsps are autologous to (derived from) the patient towhom they are administered. The hsps and/or antigenic molecules can bepurified from natural sources, chemically synthesized, or recombinantlyproduced. The invention provides methods for determining doses for humancancer immunotherapy by evaluating the optimal dose of hsp noncovalentlybound to peptide complexes in experimental tumor models andextrapolating the data. Specifically, a scaling factor not exceeding afifty fold increase over the effective dose estimated in animals, isused as the optimal prescription method for cancer immunotherapy orvaccination in human subjects.

The invention provides compositions comprising the hsp-antigenicmolecule complexes which enhance the immunocompetence of the hostindividual and elicit specific immunity against infectious agents orspecific immunity against preneoplastic and neoplastic cells. Thetherapeutic regimens and pharmaceutical compositions of the inventionare described below. These compositions are believed to have thecapacity to prevent the onset and progression of infectious diseases andprevent the development of tumor cells and to inhibit the growth andprogression of tumor cells indicating that such compositions can inducespecific immunity in infectious diseases and cancer immunotherapy.

The complexes of the invention can be used to induce an inflammatoryreaction at the tumor site and ultimately cause a regression of thetumor burden in the cancer patients treated. Cancers which can betreated with complexes of hsps noncovalently bound to exogenousantigenic molecules include, but are not limited to, human sarcomas andcarcinomas.

Accordingly, the invention provides methods of preventing and treatingcancer in an individual comprising administering a composition whichstimulates the immunocompetence of the host individual and elicitsspecific immunity against the preneoplastic and/or neoplastic cells. Asused herein, “preneoplastic” cell refers to a cell which is intransition from a normal to a neoplastic form; and morphologicalevidence, increasingly supported by molecular biologic studies,indicates that preneoplasia progresses through multiple steps.Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia,or most particularly, dysplasia (for review of such abnormal growthconditions (See Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B.Saunders Co., Philadelphia, pp. 68-79).

The therapeutic regimens and pharmaceutical compositions of theinvention may be used with additional immune response enhancers orcytokines including, but not limited to, the cytokines IFN-α, IFN-γ,IL-2, IL-4, IL-6, TNF, or other cytokine affecting immune cells. Inaccordance with this aspect of the invention, the complexes of the hspand antigenic molecule are administered in combination therapy with oneor more of these cytokines.

The invention further relates to administration of complexes ofhsp-antigenic molecules to individuals at enhanced risk of cancer due tofamilial history or environmental risk factors.

The compositions comprising hsp noncovalently bound to exogenousantigenic molecules are administered to elicit an effective specificimmune response to the complexed antigenic molecules (and not to thehsp).

In a preferred embodiment, hsp70, hsp90 and/or gp96 are noncovalentlycomplexed with exogenous antigenic molecules.

In accordance with the methods described herein, the exogenous antigenicmolecules are immunogenic or antigenic proteins or other molecules orimmunogenic/antigenic fragments or derivatives thereof. For example,exogenous antigenic molecules include but are nbt limited to differenttumor specific translatable antigens (e.g., tyrosinase, gp100, melan-A,gp75, mucins, etc.) and viral antigens including, but not limited to,proteins of immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), hepatitis type A, hepatitistype B, hepatitis type C, influenza, Varicella, adenovirus, herpessimplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest,rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papova virus, cytomegalovirus, echinovirus, arbovirus,huntavirus, coxsackie virus, mumps virus, measles virus, rubella virusand polio virus.

In a specific embodiment, antigens of cancers (e.g., tumors) orinfectious agents (e.g., viral antigen, bacterial antigens, etc.) can beobtained by purification from natural sources, by chemical synthesis, orrecombinantly, and, through in vitro procedures such as that describedbelow, noncovalently complexed to hsps.

5.1. Purification of Hsps

In an embodiment wherein the hsp portion of the hsp-antigenic moleculecomplex is desired to be purified from cells, exemplary purificationprocedures such as described in Sections 5.1.1-5.1.3 below can beemployed to purify hsp-peptide complexes, after which the hsps can bepurified from the endogenous hsp-peptide complexes in the presence ofATP or low pH, for subsequent in vitro complexing to exogenous antigenicmolecules. Although described for tumor cells, the protocols describedhereinbelow may be used to isolate hsps from any eukaryotic cells, forexample, tissues, isolated cells, or immortalized eukaryote cell linesinfected with a preselected intracellular pathogen, tumor cells or tumorcell lines.

Alternatively to isolation of native hsps from cells as described inSections 5.1.1-5.1.3, hsps can be chemically synthesized orrecombinantly produced.

5.1.1. Preparation and Purification of Hsp70-peptide Complexes

The purification of hsp70-peptide complexes has been describedpreviously, see, for example, Udono et al., 1993, J. Exp. Med.178:1391-1396. A procedure that may be used, presented by way of examplebut not limitation, is as follows:

Initially, tumor cells are suspended in 3 volumes of 1×Lysis bufferconsisting of 5 mM sodium phosphate buffer (pH 7), 150 mM NaCl, 2 mMCaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 minutes and then homogenized in a douncehomogenizer until >95% cells are lysed.

Then the lysate is centrifuged at 1000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith phosphate buffered saline (PBS) containing 2 mM Ca²⁺ and 2 mM Mg²⁺.When the cells are lysed by mechanical shearing the supernatant isdiluted with an equal volume of 2×lysis buffer prior to mixing with ConA Sepharose. The supernatant is then allowed to bind to the Con ASepharose for 2-3 hours at 4° C. The material that fails to bind isharvested and dialyzed for 36 hours (three times, 100 volumes each time)against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF.Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for20 minutes. Then the resulting supernatant is harvested and applied to aMono Q FPLC column equilibrated in 20 mM TrisAcetate pH 7.5, 20 mM NaCl,0.1 mM EDTA and 15 mM 2-mercaptoethanol. The column is then developedwith a 20 mM to 500 mM NaCl gradient and then eluted fractionsfractionated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and characterized by immunoblotting using anappropriate anti-hsp70 antibody (such as from clone N27F3-4, fromStressGen).

Fractions strongly immunoreactive with the anti-hsp70 antibody arepooled and the hsp70-peptide complexes precipitated with ammoniumsulfate; specifically with a 50%-70% ammonium sulfate cut. The resultingprecipitate is then harvested by centrifugation at 17,000 rpm (SS34Sorvall rotor) and washed with 70% ammonium sulfate. The washedprecipitate is then solubilized and any residual ammonium sulfateremoved by gel filtration on a Sephadex^(R) G25 column (Pharmacia). Ifnecessary the hsp70 preparation thus obtained can be repurified throughthe Mono Q FPCL Column as described above.

The hsp70-peptide complex can be purified to apparent homogeneity usingthis method. Typically 1 mg of hsp70-peptide complex can be purifiedfrom 1 g of cells/tissue.

The present invention further describes a new and rapid method forpurification of hsp70. This improved method uses column chromatographywith ATP affixed to a solid substratum (e.g., ATP-agarose). The hsp70yields are believed to be increased significantly and have high purity.By way of example but not limitation, purification of hsp70 byATP-agarose chromatography was carried out as follows: Meth A sarcomacells (500 million cells) were homogenized in hypotonic buffer and thelysate was centrifuged at 100,000 g for 90 minutes at 4° C. Thesupernatant was divided into two and was applied to an ADP-agarose or anATP-agarose column. The columns were washed in buffer and were elutedwith 3 mM ADP or 3 mM ATP, respectively. The eluted fractions wereanalyzed by SDS-PAGE: in both cases, apparently homogeneous preparationsof hsp70 were obtained. However, when each of the preparations wastested for presence of peptides, the ADP-bound/eluted hsp70 preparationwas found to be associated with peptides, while the ATP-bound/elutedhsp70 preparation was not.

5.1.2. Preparation and Purification of Hsp90-peptide Complexes

A procedure that can be used, presented by way of example and notlimitation, is as follows:

Initially, tumor cells are suspended in 3 volumes of 1×Lysis bufferconsisting of 5 mM sodium phosphate buffer (pH 7), 150 mM NaCl, 2 mMCaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 minutes and then homogenized in a douncehomogenizer until >95% cells are lysed.

Then the lysate is centrifuged at 1,000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cells are lysed bymechanical shearing the supernatant is diluted with an equal volume of2×lysis buffer prior to mixing with Con A Sepharose. The supernatant isthen allowed to bind to the Con A Sepharose for 2-3 hours at 4° C. Thematerial that fails to bind is harvested and dialyzed for 36 hours(three times, 100 volumes each time) against 10 mM Tris-Acetate pH 7.5,0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate is centrifuged at17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resultingsupernatant is harvested and applied to a Mono Q FPLC columnequilibrated with lysis buffer. The proteins are then eluted with a saltgradient of 200 mM to 600 mM NaCl.

The eluted fractions are fractionated by SDS-PAGE and fractionscontaining the hsp90-peptide complexes identified by immunoblottingusing an anti-hsp90 antibody such as 3G3 (Affinity Bioreagents).Hsp90-peptide complexes can be purified to apparent homogeneity usingthis procedure. Typically, 150-200 μg of hsp90-peptide complex can bepurified from 1 g of cells/tissue.

5.1.3. Preparation and Purification of gp96-peptide Complexes

A procedure that can be used, presented by way of example and notlimitation, is as follows:

A pellet of tumors is resuspended in 3 volumes of buffer consisting of30 mM sodium bicarbonate buffer (pH 7.5) and 1 mM PMSF and the cellsallowed to swell on ice 20 minutes. The cell pellet then is homogenizedin a Dounce homogenizer (the appropriate clearance of the homogenizerwill vary according to each cells type) on ice until >95% cells arelysed.

The lysate is centrifuged at 1,000 g for 10 minutes to remove unbrokencells, nuclei and other debris. The supernatant from this centrifugationstep then is recentrifuged at 100,000 g for 90 minutes. The gp96-peptidecomplex can be purified either from the 100,000 pellet or from thesupernatant.

When purified from the supernatant, the supernatant is diluted withequal volume of 2×lysis buffer and the supernatant mixed for 2-3 hoursat 4° C. with Con A Sepharose equilibrated with PBS containing 2 mM Ca²⁺and 2 mM Mg²⁺. Then, the slurry is packed into a column and washed with1×lysis buffer until the OD₂₈₀ drops to baseline. Then, the column iswashed with ⅓ column bed volume of 10% α-methyl mannoside (α-MM)dissolved in PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺, the column sealedwith a piece of parafilm, and incubated at 37° C. for 15 minutes. Thenthe column is cooled to room temperature and the parafilm removed fromthe bottom of the column. Five column volumes of the α-MM buffer areapplied to the column and the eluate analyzed by SDS-PAGE. Typically theresulting material is about 60-95% pure, however this depends upon thecell type and the tissue-to-lysis buffer ratio used. Then the sample isapplied to a Mono Q FPLC column (Pharmacia) equilibrated with a buffercontaining 5 mM sodium phosphate, pH 7. The proteins then are elutedfrom the column with a 0-1M NaCl gradient and the gp96 fraction elutesbetween 400 mM and 550 mM NaCl.

The procedure, however, may be modified by two additional steps, usedeither alone or in combination, to consistently produce apparentlyhomogeneous gp96-peptide complexes. One optional step involves anammonium sulfate precipitation prior to the Con A purification step andthe other optional step involves DEAE-Sepharose purification after theCon A purification step but before the Mono Q FPLC step.

In the first optional step, the supernatant resulting from the 100,000 gcentrifugation step is brought to a final concentration of 50% ammoniumsulfate by the addition of ammonium sulfate. The ammonium sulfate isadded slowly while gently stirring the solution in a beaker placed in atray of ice water. The solution is stirred from about ½ to 12 hours at4° C. and the resulting solution centrifuged at 6,000 rpm (Sorvall SS34rotor). The supernatant resulting from this step is removed, brought to70% ammonium sulfate saturation by the addition of ammonium sulfatesolution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor). Theresulting pellet from this step is harvested and suspended in PBScontaining 70% ammonium sulfate in order to rinse the pellet. Thismixture is centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pelletdissolved in PBS containing 2 mM Ca²⁺ and Mg²⁺. Undissolved material isremoved by a brief centrifugation at 15,000 rpm (Sorvall SS34 rotor).Then, the solution is mixed with Con A Sepharose and the procedurefollowed as before.

In the second optional step, the gp96 containing fractions eluted fromthe Con A column are pooled and the buffer exchanged for 5 mM sodiumphosphate buffer, pH 7,300 mM NaCl by dialysis, or preferably by bufferexchange on a Sephadex G25 column. After buffer exchange, the solutionis mixed with DEAE-Sepharose previously equilibrated with 5 mM sodiumphosphate buffer, pH 7, 300 mM NaCl. The protein solution and the beadsare mixed gently for 1 hour and poured into a column. Then, the columnis washed with 5 mM sodium phosphate buffer, pH 7, 300 mM NaCl, untilthe absorbance at 280 nM drops to baseline. Then, the bound protein iseluted from the column with five volumes of 5 mM sodium phosphatebuffer, pH 7, 700 mM NaCl. Protein containing fractions are pooled anddiluted with 5 mM sodium phosphate buffer, pH 7 in order to lower thesalt concentration to 175 mM. The resulting material then is applied tothe Mono Q FPLC column (Pharmacia) equilibrated with 5 mM sodiumphosphate buffer, pH 7 and the protein that binds to the Mono Q FPLCcolumn (Pharmacia) is eluted as described before.

It is appreciated, however, that one skilled in the art may assess, byroutine experimentation, the benefit of incorporating the secondoptional step into the purification protocol. In addition, it isappreciated also that the benefit of adding each of the optional stepswill depend upon the source of the starting material.

When the gp96 fraction is isolated from the 100,000 g pellet, the pelletis suspended in 5 volumes of PBS containing either 1% sodiumdeoxycholate or 1% oxtyl glucopyranoside (but without the Mg²⁺ and Ca²⁺)and incubated on ice for 1 hour. The suspension is centrifuged at 20,000g for 30 minutes and the resulting supernatant dialyzed against severalchanges of PBS (also without the Mg²⁺ and Ca²⁺) to remove the detergent.The dialysate is centrifuged at 100,000 g for 90 minutes, thesupernatant harvested, and calcium and magnesium are added to thesupernatant to give final concentrations of 2 mM, respectively. Then thesample is purified by either the unmodified or the modified method forisolating gp96-peptide complex from the 100,000 g supernatant, seeabove.

The gp96-peptide complexes can be purified to apparent homogeneity usingthis procedure. About 10-20 μg of gp96 can be isolated from 1 gcells/tissue.

5.2 Exogenous Antigenic Molecules

5.2.1. Peptides from MHC Complexes

It has been found that potentially immunogenic peptides may be elutedfrom MHC-peptide complexes using techniques well known in the art (Falk,K. et al., 1990 Nature 348:248-251; Elliott, T., et al., 1990, Nature348:195-197; Falk, K., et al., 1991, Nature 351:290-296). Once isolated,the amino acid sequence of each antigenic peptide may be determinedusing conventional amino acid sequencing methodologies. Such antigenicmolecules can then be produced by chemical synthesis or recombinantmethods, purified, and complexed to hsps in vitro.

Thus, potentially immunogenic or antigenic peptides may be isolated fromendogenous MHC-peptide complexes for use subsequently as exogenousantigenic molecules, by complexing in vitro to hsps. Exemplary protocolsfor isolating peptides and/or antigenic components from MHC complexesare set forth below in Section 5.2.1.1.

5.2.1.1. Peptides from MHC-peptide Complexes

The isolation of potentially immunogenic peptides from MHC molecules iswell known in the art and so is not described in detail herein (See,Falk, et al., 1990, Nature 348:248-251; Rotzsche, at al., 1990, Nature348:252-254; Elliott, et al., 1990, Nature 348:191-197; Falk, et al.,1991, Nature 351:290-296; Demotz, et al., 1989, Nature 343:682-684;Rotzsche, et al., 1990, Science 249:283-287), the disclosures of whichare incorporated herein by reference.

Briefly, MHC-peptide complexes may be isolated by a conventionalimmunoaffinity procedure. The peptides then may be eluted from theMHC-peptide complex by incubating the complexes in the presence of about0.1% TFA in acetonitrile. The eluted peptides may be fractionated andpurified by reverse phase HPLC, as before.

The amino acid sequences of the eluted peptides may be determined eitherby manual or automated amino acid sequencing techniques well known inthe art. Once the amino acid sequence of a potentially protectivepeptide has been determined the peptide may be synthesized in anydesired amount using conventional peptide synthesis or other protocolswell known in the art.

Peptides having the same amino acid sequence as those isolated above maybe synthesized by solid-phase peptide synthesis using procedures similarto those described by Merrifield, 1963, J. Am. Chem. Soc., 85:2149.During synthesis, N-α-protected amino acids having protected side chainsare added stepwise to a growing polypeptide chain linked by itsC-terminal and to an insoluble polymeric support i.e., polystyrenebeads. The peptides are synthesized by linking an amino group of anN-α-deprotected amino acid to an α-carboxy group of an N-α-protectedamino acid that has been activated by reacting it with a reagent such asdicyclohexylcarbodiimide. The attachment of a free amino group to theactivated carboxyl leads to peptide bond formation. The most commonlyused N-α-protecting groups include Boc which is acid labile and Fmocwhich is base labile.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe polystyrene beads. The N-α-protecting group is then removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-α-protected amino acid. The process is repeateduntil the desired peptide is synthesized. The resulting peptides arethen cleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides can be derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein (See, Atherton, etal., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRLPress, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2ndEd., Springer-Verlag).

Purification of the resulting peptides is accomplished usingconventional procedures, such as preparative HPLC using gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

5.2.2. Other Exogenous Antigenic Molecules

Antigens or antigenic portions thereof can be selected for use asantigenic molecules, for complexing to hsps, from among those known inthe art or determined by immunoassay to be able to bind to antibody orMHC molecules (antigenicity) or generate immune responses(immunogenicity). To determine immunogenicity or antigenicity bydetecting binding to antibody, various immunoassays known in the art canbe used, including but not limited to competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitin reactions, immunodiffusion assays, invivo immunoassays (using colloidal gold, enzyme or radioisotope labels,for example), western blots, immunoprecipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labelled.Many means are known in the art for detecting binding in an immunoassayand are envisioned for use. In one embodiment for detectingimmunogenicity, T cell-mediated responses can be assayed by standardmethods, e.g., in vitro cytoxicity assays or in vivo delayed-typehypersensitivity assays.

Potentially useful antigens or derivatives thereof for use as antigenicmolecules can also be identified by various criteria, such as theantigen's involvement in neutralization of a pathogen's infectivity(wherein it is desired to treat or prevent infection by such a pathogen)(Norrby, 1985, Summary, in Vaccines 85, Lerner, et al. (eds.), ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 388-389), typeor group specificity, recognition by patients' antisera or immune cells,and/or the demonstration of protective effects of antisera or immunecells specific for the antigen. In addition, where it is desired totreat or prevent a disease caused by pathogen, the antigen's encodedepitope should preferably display a small or no degree of antigenicvariation in time or amongst different isolates of the same pathogen.

Preferably, where it is desired to treat or prevent cancer, knowntumor-specific antigens or fragments or derivatives thereof are used.For example, such tumor specific or tumor-associated antigens includebut are not limited to KS 1/4 pan-carcinoma antigen (Perez and Walker,1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415);ovarian carcinoma antigen (CA125) (Yu, et al., 1991, Cancer Res.51(2):468-475); prostatic acid phosphate (Tailer, et al., 1990, Nucl.Acids Res. 18(16):4928); prostate specific antigen (Henttu and Vihko,1989, Biochem. Biophys. Res. Comm. 160(2):903-910; Israeli, et al.,1993, Cancer Res. 53:227-230); melanoma-associated antigen p97 (Estin,et al., 1989, J. Natl. Cancer Inst. 81(6):445-446); melanoma antigengp75 (Vijayasardahl, et al., 1990, J. Exp. Med. 171(4):1375-1380); highmolecular weight melanoma antigen (Natali, et al., 1987, Cancer59:55-63) and prostate specific membrane antigen.

In a specific embodiment, an antigen or fragment or derivative thereofspecific to a certain tumor is selected for complexing to hsp andsubsequent administration to a patient having that tumor.

Preferably, where it is desired to treat or prevent viral diseases,molecules comprising epitopes of known viruses are used. For example,such molecules comprise epitopes from proteins of viruses including, butnot limited to, hepatitis type A hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial. virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

Preferably, where it is desired to treat or prevent bacterialinfections, molecules comprising epitopes of known bacteria are used.For example, such antigenic epitopes may be from bacteria including, butnot limited to, mycobacteria rickettsia, mycoplasma, neisseria andlegionella.

Preferably, where it is desired to treat or prevent protozoalinfectious, molecules comprising epitopes of known protozoa are used.For example, such protozoa include, but are not limited to, leishmania,kokzidioa, and trypanosoma.

Preferably, where it is desired to treat or prevent parasiticinfectious, molecules comprising epitopes of known parasites are used.For example, such antigenic epitopes may be from parasites including,but not limited to, chlamydia and rickettsia.

5.3. In Vitro Production of Stress Protein-antigenic Molecule Complexes

As will be appreciated by those skilled in the art, exogenous antigenicmolecules, either purified from natural sources or chemicallysynthesized or recombinantly produced, may be reconstituted with avariety of naturally purified or chemically synthesized or recombinantlyproduced stress proteins in vitro to generate immunogenic noncovalentstress protein-antigenic molecule complexes. A preferred, exemplaryprotocol for noncovalently complexing a stress protein and an exogenousantigenic molecule in vitro is described below.

Prior to complexing, the hsps are pretreated with ATP or low pH toremove any peptides that may be associated with the hsp of interest.When the ATP procedure is used, excess ATP is removed from thepreparation by the addition of apyranase as described by Levy, et al.,1991, Cell 67:265-274. When the low pH procedure is used, the buffer isreadjusted to neutral pH by the addition of pH modifying reagents.

The antigenic molecules (1 μg) and the pretreated hsp (9 μg) are admixedto give an approximately 5 antigenic molecule:1 stress protein molarratio. Then, the mixture is incubated for 15 minutes to 3 hours at roomtemperature in a suitable binding buffer such as one containing 20 mMsodium phosphate, pH 7.2, 350 mM NaCl, 3 mM MgCl₂ and 1 mM phenyl methylsulfonyl fluoride (PMSF). The preparations are centrifuged throughCentricon 10 assembly (Millipore) to remove any unbound peptide. Theassociation of the peptides with the stress proteins can be assayed bySDS-PAGE. This is the preferred method for in vitro complexing ofpeptides isolated from MHC-peptide complexes of peptides disassociatedfrom endogenous hsp-peptide complexes.

In an alternative embodiment of the invention, preferred for producingcomplexes of hsps70 to exogenous antigenic molecules that are proteins,5-10 micrograms of purified hsp is incubated with equimolar quantitiesof the antigenic molecule in 20 mM sodium phosphate buffer pH 7.5, 0.5MNaCl, 3 mM MgCl₂ and 1 mM ADP in a volume of 100 microliter at 37° C.for 1 hr. This incubation mixture is further diluted to 1 ml inphosphate-buffered saline.

In an alternate embodiment of the invention, preferred for producingcomplexes of gp96 or hsp90 to peptides 5-10 micrograms of purified gp96or hsp90 is incubated with equimolar or excess quantities of theantigenic peptide in a suitable buffer such as one containing 20 mMsodium phosphate buffer 7.5, 0.5 m NaCl, 3 mM MgCl₂ at 60-65° C. for5-20 minutes. This incubation mixture is allowed to cool to roomtemperature and centrifuged more than once if necessary throughCentrican 10 assembly (Millipore to remove any unbound peptide).

Following complexing, the immunogenic stress protein-antigenic moleculecomplexes can optionally be assayed in vitro using for example the mixedlymphocyte target cell assay (MLTC) described below. Once immunogeniccomplexes have been isolated they can be optionally characterizedfurther in animal models using the preferred administration protocolsand excipients discussed below.

5.4. Determination of Immunogenicity of Stress Protein-peptide Complexes

In an optional procedure, the purified stress protein-antigenic moleculecomplexes can be assayed for immunogenicity using the mixed lymphocytetarget culture assay (MLTC) well known in the art.

By way of example but not limitation, the following procedure can beused. Briefly, mice are injected subcutaneously with the candidatestress protein-antigenic molecule complexes. Other mice are injectedwith either other stress protein peptide complexes or whole infectedcells which act as positive controls for the assay. The mice areinjected twice, 7-10 days apart. Ten days after the last immunization,the spleens are removed and the lymphocytes released. The releasedlymphocytes may be restimulated subsequently in vitro by the addition ofdead cells that expressed the complex of interest.

For example, 8×10⁶ immune spleen cells may be stimulated with 4×10⁴mitomycin C treated or γ-irradiated (5-10,000 rads) infected cells (orcells transfected with an appropriate gene, as the case may be) in 3 mlRPMI medium containing 10% fetal calf serum. In certain cases 33%secondary mixed lymphocyte culture supernatant may be included in theculture medium as a source of T cell growth factors (See, Glasebrook, etal., 1980, J. Exp. Med. 151:876). To test the primary cytotoxic T cellresponse after immunization, spleen cells may be cultured withoutstimulation. In some experiments spleen cells of the immunized mice mayalso be restimulated with antigenically distinct cells, to determine thespecificity of the cytotoxic T cell response.

Six days later the cultures are tested for cytotoxicity in a 4 hour⁵¹Cr-release assay (See, Palladino, et al., 1987, Cancer Res.47:5074-5079 and Blachere, at al., 1993, J. Immunotherapy 14:352-356).In this assay, the mixed lymphocyte culture is added to a target cellsuspension to give different effector:target (E:T) ratios (usually 1:1to 40:1). The target cells are prelabelled by incubating 1×10⁶ targetcells in culture mediume containing 200 mCi ⁵¹Cr/ml for one hour at 37°C. The cells are washed three times following labeling. Each assay point(E:T ratio) is performed in triplicate and the appropriate controlsincorporated to measure spontaneous ⁵¹Cr release (no lymphocytes addedto assay) and 100% release (cells lysed with detergent). Afterincubating the cell mixtures for 4 hours, the cells are peletted bycentrifugation at 200 g for 5 minutes. The amount of ⁵¹Cr released intothe supernatant is measured by a gamma counter. The percent cytotoxicityis measured as cpm in the test sample minus spontaneously released cpmdivided by the total detergent released cpm minus spontaneously releasedcpm.

In order to block the MHC class I cascade a concentrated hybridomasupernatant derived from K-44 hybridoma cells (an anti-MHC class Ihybridoma) is added to the test samples to a final concentration of12.5%.

5.5. Formulation

Complexes of the invention (of hsps noncovalently bound to exogenousantigenic molecules) may be formulated into pharmaceutical preparationsfor administration to mammals for treatment of tumors and infectiousdiseases. Preferred dosages, routes of administration and therapeuticregimens are described in copending application by P. Srivastavaentitled “Compositions and Methods for the Prevention and Treatment ofPrimary and Metastatic Neoplastic Diseases and Infectious Diseases withHeat Shock/Stress Proteins, filed on even date herewith, which isincorporated by reference herein in its entirety. For example,pharmaceutical formulations are provided, based on a newly-discoveredextrapolation and scaling of animal dosage to human, comprisingcompositions of complexes of antigenic molecules and heat shock/stressproteins, including but not limited to hsp70, hsp90, gp96 either aloneor in combination. Specifically, interspecies dose-response equivalencefor hsp noncovalently bound to antigenic molecules for a human dose isestimated as the product of the therapeutic dosage observed in mice anda single scaling ratio, not exceeding a 50-fold increase. In preferredaspects, an amount of hsp70- and/or gp96-antigenic molecule complex isadministered to a human that is in the range of about 10-600 μg,preferably 10-100 μg, most preferably about 25 μg, given once weekly forabout 4-6 weeks, subcutaneously with the site of administration variedsequentially. Preferred amounts for hsp90-antigenic molecule complexesare in the range of 50-5,000 μg, preferably 100 μg.

Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may be prepared, packaged, andlabelled for treatment of the indicated tumor, such as human sarcomasand carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastqma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenström's macroglobulinemia, and heavychain disease. Alternatively, it can be formulated and labeled fortreatment of the appropriate infectious disease.

If the complex is water-soluble, then it may be formulated in anappropriate buffer, for example, phosphate buffered saline or otherphysiologically compatible solutions. Alternatively, if the resultingcomplex has poor solubility in aqueous solvents, then it may beformulated with a non-ionic surfactant such as Tween, or polyethyleneglycol. Thus, the compounds and their physiologically acceptablesolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral, rectal administration or, in the case of tumors, directlyinjected into a solid tumor.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well known examplesof delivery vehicles or carriers for hydrophilic drugs.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically or prophylactically effective amounts of thehsp-antigenic molecule complexes in pharmaceutically acceptable form.The hsp-antigenic molecule complex in a vial of a kit of the inventionmay be in the form of a pharmaceutically acceptable solution, e.g., incombination with sterile saline, dextrose solution, or bufferedsolution, or other pharmaceutically acceptable sterile fluid.Alternatively, the complex may be lyophilized or desiccated; in thisinstance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomplex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of hsp-antigenic molecule complexes by aclinician or by the patient.

5.6. Target Infectious Diseases

Infectious diseases that can be treated or prevented by the methods ofthe present invention are caused by infectious agents including, but notlimited to viruses, bacteria, fungi protozoa and parasites.

Viral diseases that can be treated or prevented by the methods of thepresent invention include, but are not limited to, those caused byhepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

Bacterial diseases that can be treated or prevented by the methods ofthe present invention are caused by bacteria including, but not limitedto, mycobacteria rickettsia, mycoplasma, neisseria and legionella.

Protozoal diseases that can be treated or prevented by the methods ofthe present invention are caused by protozoa including, but not limitedto, leishmania, kokzidioa, and trypanosoma.

Parasitic diseases that can be treated or prevented by the methods ofthe present invention are caused by parasites including, but not limitedto, chlamydia and rickettsia.

5.7. Target Cancers

Cancers that can be treated or prevented by the methods of the presentinvention include, but not limited to human sarcomas and carcinomas,e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenström's macroglobulinemia, and heavychain disease. Specific examples of such cancers are described in thesections below.

In a specific embodiment the cancer is metastatic. In another specificembodiment, the patient having a cancer is immunosuppressed by reason ofhaving undergone anti-cancer therapy (e.g., chemotherapy radiation)prior to administration of the hsp-antigenic molecule complexes of theinvention. In another specific embodiment, the cancer is a tumor.

5.7.1. Colorectal Cancer Metastatic to the Liver

In 1992, approximately 150,000 Americans were diagnosed with colorectalcancer and more than 60,000 died as a result of colorectal metastases.At the time of their deaths, 80 percent of patients with colorectalcancer have metastatic disease involving the liver, and one-half ofthese patients have no evidence of other (extrahepatic) metastases. Mostmetastatic tumors of the liver are from gastrointestinal primaries.Unfortunately, the natural history of metastatic liver lesions carries agrave prognosis and systemic chemotherapy regimens have been unable toinduce significant response rates or alter length of survival (Drebin,J. A., et al., in Current Therapy In Oncology, ed. J. E. Niederhuber, B.C. Decker, Mosby, 1993, p.426).

Colorectal cancer initially spreads to regional lymph nodes and thenthrough the portal venous circulation to the liver, which represents themost common visceral site of metastasis. The symptoms that lead patientswith colorectal cancer to seek medical care vary with the anatomicallocation of the lesion. For example, lesions in the ascending colonfrequency ulcerate, which leads to chronic blood loss in the stool.

Radical resection offers the greatest potential for cure in patientswith invasive colorectal cancer. Before surgery, the CEA titer isdetermined. Radiation therapy and chemotherapy are used in patients withadvanced colorectal cancer. Results with chemotherapeutic agents (e.g.,5-fluorouracil) are mixed and fewer than 25 percent of patientsexperience a greater than 50 percent reduction in tumor mass (Richards,2d., F., et al., 1986, J. Clin. Oncol. 4:565).

Patients with widespread metastases have limited survival and systemicchemotherapy has little impact in this group of patients. In addition,systemically administered chemotherapy is often limited by the severityof toxicities associated with the various agents, such as severediarrhea, mucositis and/or myelosuppression. Other techniques, includinghepatic radiation, systemic chemotherapy, hepatic arterial ligation,tumor embolization and immunotherapy have all been explored, but, forthe most part, have proven ineffectual in prolonging patient survival.

In a specific embodiment, the present invention provides compositionsand methods for enhancing tumor specific immunity in individualssuffering from colorectal cancer metastasized to the liver, in order toinhibit the progression of the neoplastic disease.

Accordingly, as an example of the method of the invention, gp96 isadministered to a patient diagnosed with colorectal cancer, with orwithout liver metastasis, via one of many different routes ofadministration, the preferred routes being subcutaneous at differentanatomical sites, e.g., left arm, right arm, left belly, right belly,left thigh, right thigh, etc. These routes of administration are used insequence and the site of injection is varied for each weekly injectionas described in Section 7. The preparations and use of therapeuticallyeffective compositions for the prevention and treatment of primary andmetastatic cancers are described in detail in the sections which followand by way of example, infra.

5.7.2. Hepatocellular Carcinoma

Hepatocellular carcinoma is generally a disease of the elderly in theUnited States. Although many factors may lead to hepatocellularcarcinoma, the disease is usually limited to those persons withpreexisting liver disease. Approximately 60 to 80 percent of patients inthe United States with hepatocellular carcinoma have a cirrhotic liverand about four percent of individuals with a cirrhotic liver eventuallydevelop hepatocellular carcinoma (Niederhuber, J. E., (ed.), 1993,Current Therapy in Oncology, B. C. Decker, Mosby). The risk is highestin patients whose liver disease is caused by inherited hemochromatosisor hepatic B viral infection (Bradbear, R. A., et al., 1985, J. Natl.Cancer Inst. 75:81; Beasley, R. P., et al., 1981, Lancet 2:1129). Othercauses of cirrhosis that can lead to hepatocellular carcinoma includealcohol abuse and hepatic fibrosis caused by chronic administration ofmethotrexate. The most frequent symptoms of hepatocellular carcinoma arethe development of a painful mass in the right upper quadrant orepigastrium, accompanied by weight loss. In patients with cirrhosis, thedevelopment of hepatocellular carcinoma is preceded by ascites, portalhypertension and relatively abrupt clinical deterioration. In mostcases, abnormal values in standard liver function tests such as serumaminotransferase and alkaline phosphatase are observed.

CT scans of the liver are used to determine the anatomic distribution ofhepatocellular carcinoma and also provide orientation for percutaneousneedle biopsy. Approximately 70 percent of patients with hepatocellularcarcinoma have an elevated serum alpha-fetoprotein concentration(McIntire, K. R., et al., 1975, Cancer Res. 35:991) and itsconcentration correlates with the extent of the disease.

Radical resection offers the only hope for cure in patients withhepatocellular carcinoma. Such operative procedures are associated withfive-year survival rates of 12 to 30 percent. Liver transplantation mayimprove survival of some younger individuals. However, most patients arenot surgical candidates because of extensive cirrhosis multifocal tumorpattern or scarcity of compatible donor organs. Chemotherapeutic agentshave been administered either by intravenous route or through anintrahepatic arterial catheter. Such therapy has sometimes been combinedwith irradiation to the liver. Reductions in the size of measurabletumors of 50% or more have been reported in some patients treated witheither systemic doxorubicin or 5-fluorouracil. However, chemotherapyoften induces immunosuppression and rarely causes the tumor to disappearcompletely and the duration of response is short. The prognosis forpatients with hepatocellular carcinoma is negatively correlated withcirrhosis and metastases to the lungs or bone. Median survival forpatients is only four to six months. In another specific embodiment, thepresent invention provides compositions and methods for enhancingspecific immunity in individuals suffering from hepatocellular carcinomain order to inhibit the progression of the neoplastic disease andultimately irradiate all preneoplastic an neoplastic cells.

5.7.3. Breast Cancer

Another specific aspect of the invention relates to the treatment ofbreast cancer. The American Cancer Society estimated that in 1992180,000 American women were diagnosed with breast cancer and 46,000succumbed to the disease (Niederhuber, J. E.ed. Current Therapy inOncology B. C. Decker, Mosby, 1993). This makes breast cancer the secondmajor cause of cancer death in women, ranking just behind lung cancer. Adisturbing fact is the observation that breast cancer has beenincreasing at a rate of 3 percent per year since 1980 (Niederhuber, J.E., ed. Current Therapy in Oncology, B. C. Decker, Mosby, (1993)). Thetreatment of breast cancer presently involves surgery, radiation,hormonal therapy and/or chemotherapy. Consideration of two breast cancercharacteristics, hormone receptors and disease extent, has governed howhormonal therapies and standard-dose chemotherapy are sequenced toimprove survival and maintain or improve quality of life. A wide rangeof multidrug regimens have been used as adjuvant therapy in breastcancer patients, including, but not limited to combinations of 2cyclophosphamide, doxorubicin, vincristine methotrexate, 5-fluorouraciland/or leucovorin. In a specific embodiment, the present inventionprovides hsp compositions and methods for enhancing specific immunity topreneoplastic and neoplastic mammary cells in women. The presentinvention also provides compositions and methods for preventing thedevelopment of neoplastic cells in women at enhanced risk for breastcancer, and for inhibiting cancer cell proliferation and metastasis.These compositions can be applied alone or in combination with eachother or with biological response modifiers.

5.8. Prevention and Treatment of Primary and Metastatic NeoplasticDiseases

There are many reasons why immunotherapy as provided by the presentinvention is desired for use in cancer patients. First, if cancerpatients are immunosuppressed and surgery, with anesthesia, andsubsequent chemotherapy, may worsen the immunosuppression, then withappropriate immunotherapy in the preoperative period, thisimmunosuppression may be prevented or reversed. This could lead to fewerinfectious complications and to accelerated wound healing. Second, tumorbulk is minimal following surgery and immunotherapy is most likely to beeffective in this situation. A third reason is the possibility thattumor cells are shed into the circulation at surgery and effectiveimmunotherapy applied at this time can eliminate these cells.

In a specific embodiment, the preventive and therapeutic methods of theinvention are directed at enhancing the immunocompetence of the cancerpatient either before surgery, at or after surgery, and to inducetumor-specific immunity to cancer cells, with the objective beinginhibition of cancer, and with the ultimate clinical objective beingtotal cancer regression and eradication.

5.9. Monitoring of Effects During Cancer Prevention and Immunotherapywith Hsp-peptide Complexes

The effect of immunotherapy with hsp-antigenic molecule complexes ondevelopment and progression of neoplastic diseases can be monitored byany methods known to one skilled in the art, including but not limitedto measuring: a) delayed hypersensitivity as an assessment of cellularimmunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels oftumor specific antigens, e.g., carcinoembryonic (CEA) antigens; d)changes in the morphology of tumors using techniques such as a computedtomographic (CT) scan; e) changes in levels of putative biomarkers ofrisk for a particular cancer in individuals at high risk, and f) changesin the morphology of tumors using a sonogram.

5.9.1. Delayed Hypersensitivity Skin Test

Delayed hypersensitivity skin tests are of great value in the overallimmunocompetence and cellular immunity to an antigen. Inability to reactto a battery of common skin antigens is termed anergy (Sato, T., et al,1995, Clin. Immunol. Pathol. 74:35-43).

Proper technique of skin testing requires that the antigens be storedsterile at 4° C., protected from light and reconstituted shorted beforeuse. A 25- or 27-gauge need ensures intradermal, rather thansubcutaneous, administration of antigen. Twenty-four and 48 hours afterintradermal administration of the antigen, the largest dimensions ofboth erythema and induration are measured with a ruler. Hypoactivity toany given antigen or group of antigens is confirmed by testing withhigher concentrations of antigen or, in ambiguous circumstances, by arepeat test with an intermediate test.

5.9.2. Activity of Cytolytic T-lymphocytes In Vitro

8×10⁶ Peripheral blood derived T lymphocytes isolated by theFicoll-Hypaque centrifigation gradient technique, are restimulated with4×10⁴ mitomycin C treated tumor cells in 3 ml RPMI medium containing 10%fetal calf serum. In some experiments, 33% secondary mixed lymphocyteculture supernatant or IL-2, is included in the culture medium as asource of T cell growth factors.

In order to measure the primary response of cytolytic T-lymphocytesafter immunization, T cells are cultured without the stimulator tumorcells. In other experiments, T cells are restimulated with antigenicallydistinct cells. After six days, the cultures are tested for cytotoxityin a 4 hour ⁵¹Cr-release assay. The spontaneous ⁵¹Cr-release of thetargets should reach a level less than 20%. For the anti-MHC class Iblocking activity, a tenfold concentrated supernatant of W6/32 hybridomais added to the test at a final concentration of 12.5% (Heike M., etal., J. Immunotherapy 15:165-174).

5.9.3. Levels of Tumor Specific Antigens

Although it may not be possible to detect unique tumor antigens on alltumors, many tumors display antigens that distinguish them from normalcells. The monoclonal antibody reagents have permitted the isolation andbiochemical characterization of the antigens and have been invaluablediagnostically for distinction of transformed from nontransformed cellsand for definition of the cell lineage of transformed cells. Thebest-characterized human tumor-associated antigens are the oncofetalantigens. These antigens are expressed during embryogenesis, but areabsent or very difficult to detect in normal adult tissue. The prototypeantigen is carcinoembryonic antigen (CEA), a glycoprotein found on fetalgut an human colon cancer cells, but not on normal adult colon cells.Since CEA is shed from colon carcinoma cells and found in the serum, itwas originally thought that the presence of this antigen in the serumcould be used to screen patients for colon cancer. However, patientswith other tumors, such as pancreatic and breast cancer, also haveelevated serum levels of CEA. Therefore, monitoring the fall and rise ofCEA levels in cancer patients undergoing therapy has proven useful forpredicting tumor progression and responses to treatment.

Several other oncofetal antigens have been useful for diagnosing andmonitoring human tumors, e.g., alpha-fetoprotein, an alpha-globulinnormally secreted by fetal liver and yolk sac cells, is found in theserum of patients with liver and germinal cell tumors and can be used asa matter of disease status.

5.9.4. Computed Tomographic (CT) Scan

CT remains the choice of techniques for the accurate staging of cancers.CT has proved more sensitive and specific than any other imagingtechniques for the detection of metastases.

5.9.5. Measurement of Putative Biomarkers

The levels of a putative biomarker for risk of a specific cancer aremeasured to monitor the effect of hsp noncovalently bound to peptidecomplexes. For example, in individuals at enhanced risk for prostatecancer, serum prostate-specific antigen (PSA; is measured by theprocedure described by Brawer, M. K., et. al., 1992, J. Urol.147:841-845, and Catalona, W. J., et al., 1993, JAMA 270:948-958; or inindividuals at risk for colorectal cancer CEA is measured as describedabove in Section 4.5.3; and in individuals at enhanced risk for breastcancer, 16-α-hydroxylation of estradiol is measured by the proceduredescribed by Schneider, J. et al., 1982, Proc. Natl. Acad. Sci. USA79:3047-3051. The references cited above are incorporated by referenceherein in their entirety.

5.9.6. Sonogram

A sonogram remains an alternative choice for technique for the accuratestaging of cancers.

6. EXAMPLE Administration of hsp-exogenous Antigens in the Treatment ofHepatocellular Carcinoma

Patients with hepatocellular carcinoma are injected with hsp-antigenicmolecule peptide complexes prepared in vitro from purified hsp andpurified antigen. The antigen used is carcinoembryonic antigen (CEA).Treatment with hsp-antigen complexes is started any time after surgery.However, if the patient has received chemotherapy, hsp-antigen complexesare usually administered after an interval of four weeks or more so asto allow the immune system to recover. The immunocompetence of thepatient is tested by procedures described in sections 5.9 above.

The therapeutic regimen includes weekly injections of the hsp-antigencomplex, dissolved in saline or other physiologically compatiblesolution.

The dosage used for hsp70 or gp96 is in the range of 10-600 micrograms,with the preferred dosage being 10-100 micrograms. The dosage used forhsp90 is in the range of 50 to 5,000 micrograms, with the preferreddosage being about 100 micrograms.

The route and site of injection is varied each time, for example, thefirst injection is given subcutaneously on the left arm, the secondinjection on the right arm, the third injection on the left abdominalregion, the fourth injection on the right abdominal region, the fifthinjection on the left thigh, the sixth injection on the right thigh,etc. The same site is repeated after a gap of one or more injections. Inaddition, injections are split and each half of the dose is administeredat a different site on the same day.

Overall, the first four to six injections are given at weekly intervals.Subsequently, two injections are given at two-week intervals, followedby a regimen of injections at monthly intervals. The effect ofhsp-antigen complexes therapy is monitored by measuring: a) delayedhypersensitivity as an assessment of cellular immunity; b) activity ofcytolytic T-lymphocytes in vitro; c) levels of tumor specific antigens,e.g., carcinoembryonic (CEA) antigens; d) changes in the morphology oftumors using techniques such as a computed tomographic (CT) scan; and e)changes in putative biomarkers of risk for a particular cancer inindividuals at high risk.

Depending on the results obtained, the therapeutic regimen is developedto maintain and/or boost the immunological responses of the patient,with the ultimate goal of achieving tumor regression and completeeradication of cancer cells.

7. EXAMPLE Administration of hsp-exogenous Antigen Complexes in theTreatment of Colorectal Cancer

Hsp-antigen complexes (comprising gp96, hsp70, hsp90 or a combinationthereof) are administered as adjuvant therapy and as prophylacticadjuvant therapy in patients after complete reduction of colorectalcancer to eliminate undetectable micrometastases and to improvesurvival.

The therapeutic and prophylactic regimens used in patients sufferingfrom colorectal cancer are the same as those described in Section 6above for patients recovering with hepatocellular carcinoma. The antigenused as the exogenous antigenic molecule is carcinoembryonic antigen.The methods of monitoring of patients under clinical evaluation forprevention and treatment of colorectal cancer is done by proceduresdescribed in Section 5.9.

8. EXAMPLE Induction of CTL-response to hsp70-ovalbumin Complex

8.1. Materials and Methods

Hsp70-ovalbumin complex was prepared in vitro. Briefly, 5-10 microgramsof purified hsp70 was incubated with equimolar quantities of ovalbuminin 20 mM sodium phosphate buffer pH 7.5, 0.5 NaCl, 3 mM MgCl₂ and 1 mMADP in a volume of 100 microliter at 37° C. for 1 hour. This incubationmixture was then further diluted to 1 ml in phosphate-buffered salineand injected sub-cutaneously into the mammal of choice, the C57BL/6strain of mice.

The injections were repeated once a week interval. The hsp70-ovalbumincomplex was prepared fresh for each injection. A total of two injectionswas administered before sacrificing the animals. Two mice in each groupwere immunized with: a) control vehicle; b) ovalbumin alone; c) hsp70alone; or d) hsp70-ovalbumin complex.

T cells were isolated from the spleen of each mouse using the Tcell-gradient centrifugation technique and 8×10⁵ T cells were incubatedwith 4×10⁴ EG7 cells (positive for ovalbumin antigen) or EL4 cells(negative for ovalbumin antigen). The CTL response was measured as %⁵¹Crrelease.

8.2. Results

Hsp70-ovalbumin complex induced a far greater CTL response thanovalbumin alone or hsp70 alone (FIG. 1A). However, the T cells did notrespond to the EL4 cells which lack the ovalbumin antigen (FIG. 1B).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method of eliciting an immune response againstan antigenic peptide or protein in an individual comprisingadministering to the individual a complex of a heat shock proteinnoncovalently bound to the antigenic peptide or protein, wherein thecomplex is made in vitro, and wherein the antigenic peptide or proteinis not isolated from a heat shock protein-antigenic peptide or proteincomplex from within a cell.
 2. The method according to claim 1 whereinthe antigenic peptide or protein is selected from the group consistingof a cancer antigen, an antigen of an infectious agent, and a moleculedisplaying antigenicity of a cancer antigen or antigen of an infectiousagent.
 3. The method according to claim 2 wherein the antigenic peptideor protein is a cancer antigen from a cancer selected from the groupconsisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiplemyeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. 4.The method according to claim 2 wherein the antigenic peptide or proteinis an antigen of an infectious agent selected from the group consistingof a virus, bacterium, protozoan, and parasite.
 5. The method accordingto claim 4 wherein the infectious agent is a virus selected from thegroup consisting of hepatitis type A, hepatitis type B, hepatitis typeC, influenza, varicella, adenovirus, herpes simplex type I (HSV-I),herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,rotavirus, respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).
 6. The method according to claim 4 wherein theinfectious agent is a bacterium selected from the group consisting ofmycobacteria, rickettsia, mycoplasma, neisseria and legionella.
 7. Themethod according to claim 4 wherein the infectious agent is a protozoanselected from the group consisting of leishmania, kokzidioa, andtrypanosoma.
 8. The method according to claim 4 wherein the infectiousagent is a parasite selected from the group consisting of chlamydia andrickettsia.
 9. The method according to claim 1 wherein the heat shockprotein is selected from the group consisting of hsp70, hsp90, and gp96.10. The method according to claim 1 wherein the heat shock protein ishsp70.
 11. The method according to claim 1 wherein the heat shockprotein is hsp90.
 12. The method according to claim 1 wherein the heatshock protein is gp96.
 13. The method according to claim 1 wherein theindividual is a human.
 14. The method according to claim 1 wherein theantigenic peptide or protein is a tumor-specific antigen, atumor-associated antigen, or an antigenic fragment of a tumor-specificantigen or tumor-associated antigen.
 15. The method according to claim 1wherein the complex comprises a population of complexes wherein the heatshock protein is a combination of hsp70 and hsp90, hsp70 and gp96, hsp90and gp96, or hsp70, hsp90, and gp96.